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In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H+/peptide cotransporters. L-4,4′-Biphenylalanyl–L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H+/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H+/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H+/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [14C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (Ki = 24 μM and 3.4 μM, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[3H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and δ-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis–Menten constant (Kt) of 7.6 μM and a maximal velocity (Vmax) of 1.1 nmol·30 min−1·mg of protein−1. Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[3H]Pro is a valuable substrate for both mechanistic and structural studies of H+/peptide transporter proteins.